Catalyst-free surface functionalization and polymer grafting

ABSTRACT

The present application relates to a method for preparing a functionalized surface and the use in DNA sequencing and other diagnostic applications. A substrate with a surface comprising a silane or a silane derivative covalently attached to optionally substituted cycloalkene or optionally substituted heterocycloalkene can be used for direct conjugation with a functionalized molecule of interest, such as a polymer, a hydrogel, an amino acid, a nucleoside, a nucleotide, a peptide, a polynucleotide, or a protein. In some embodiments, the silane or silane derivative contains optionally substituted norbornene or norbornene derivatives.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/316,478, filed Jun. 26, 2014, to be issued as U.S. Pat. No. 9,994,687on Jun. 12, 2018, which claims the benefit of priority to U.S.Provisional Patent Application No. 61/841,647 filed on Jul. 1, 2013, andU.S. Provisional Patent Application No. 61/971,381 filed on Mar. 27,2014, all of which are hereby expressly incorporated by reference intheir entireties.

FIELD

In general, the present application relates to the fields of chemistry,biology and material science. More specifically, the present applicationrelates to a substrate with a surface comprising a silane or silanederivative comprising optionally substituted unsaturated moietiescomprising cycloalkenes, cycloalkynes, heterocycloalkenes, orheterocycloalkynes covalently attached thereto for direct conjugationwith a functionalized molecule of interest. Methods for preparing afunctionalized surface and the use in DNA sequencing and otherdiagnostic applications are also disclosed.

BACKGROUND

Polymer or hydrogel-coated substrates are used in many technologicalapplications. For example, implantable medical devices can be coatedwith biologically inert polymers. In another example, polymer orhydrogel coated substrates are used for the preparation and/or analysisof biological molecules. Molecular analyses, such as certain nucleicacid sequencing methods, rely on the attachment of nucleic acid strandsto a polymer or hydrogel-coated surface of a substrate. The sequences ofthe attached nucleic acid strands can then be determined by a number ofdifferent methods that are well known in the art.

In certain sequencing-by-synthesis processes, one or more surfaces of aflow cell are coated with a polymer or a hydrogel to which nucleic acidsare attached. Current commercial flow cells utilize a non-attached gelcoating. Use of an appropriate conjugating chemistry may provide forcommercially viable flow cells having covalently attached gel coatings.Considerations such as cost of materials, compatibility withmanufacturing processes, stability during storage and shipping, and theability to support downstream chemical processing steps such as nucleicacid amplification and sequencing are important to consider. Thisdisclosure provides a particularly useful chemistry having severaladvantages as will become apparent from the disclosure.

SUMMARY

The present application discloses new ways to prepare the surface of asubstrate for direct conjugation of an appropriately functionalizedhydrogel, polymer, molecule or biomolecule of interest. The surface istreated with a silane or a silane derivative comprising a firstplurality of optionally substituted unsaturated moieties selected fromcycloalkenes, cycloalkynes, heterocycloalkenes, or heterocycloalkynescovalently bounded to the silicon atoms of the silane or silanederivative either directly or via linkers, such as norbornene,cyclooctene, cyclooctyne, bicycloalkynes, or any cycloalkenes,cycloalkynes, heterocycloalkenes, heterocycloalkynes or derivativesthereof where ring strain is present, without the need of catalyst oradditional cross-linking agents. In particular embodiments, theimplementation of the present application eliminates the use ofadditional cross-linking compounds or catalysts, and provides a singlesurface modification process as a common starting point to obtain alarge variety of functionalized surfaces for use in DNA sequencing andother diagnostic applications. In addition, substrate surfaces preparedaccordingly to the present application were found to have higherstability resulting in longer shelf life and reduced surfacecontamination upon storage. Lastly, substrate surfaces preparedaccording to the present application were found to have unique surfaceaffinity compared to standard silanes (such as APTES or APTMS), whichresulted in better wettability with aqueous based components and morehomogeneous coatings.

The present application also discloses new ways to graft primers to thesurface of a substrate. In one embodiment, the surface is treated withsilane or a silane derivative comprising a first plurality of optionallysubstituted unsaturated moieties selected from cycloalkenes,cycloalkynes, heterocycloalkenes, or heterocycloalkynes covalentlybounded to the silicon atoms of the silane or silane derivative eitherdirectly or via linkers, without the need of catalyst or additionalcross-linking agents. Then, the primer is pre-conjugated with afunctional molecule with functional groups covalently bonded tooligonucleotides, where the oligonucleotides comprises a secondplurality of optionally substituted unsaturated moieties selected fromcycloalkenes, cycloalkynes, heterocycloalkenes, or heterocycloalkynes,such as cyclooctyne or bicycloalkynes, e.g., bicyclo[6.1.0]non-4-yne.Finally, the pre-conjugated primer is covalently attached to the silaneor silane derivative by reacting the functional groups of thefunctionalized molecule with the unsaturated moieties of the silane orsilane derivative.

Some embodiments described herein relate to a substrate comprising afirst surface comprising silane or a silane derivative covalently boundto a functionalized molecule through a first plurality of unsaturatedmoieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants or combinationsthereof covalently attached to silicon atoms of the silane or silanederivative. In some embodiments, the substrate further comprisesoligonucleotides covalently attached to the functionalized moleculethrough a second plurality of unsaturated moieties selected fromcycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, oroptionally substituted variants or combinations thereof.

Some embodiments described herein relate to a method of immobilizing afunctionalized molecule comprising functional groups to a first surfaceof a substrate, the method comprising: applying silane or a silanederivative comprising a first plurality of unsaturated moieties selectedfrom cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes,or optionally substituted variants or combinations thereof covalentlyattached thereto onto the first surface of the substrate; and covalentlyattaching the functionalized molecule to the silane or silane derivativeby reacting the functional groups of the functionalized molecule withthe first plurality of unsaturated moieties to form a coating layer. Insome embodiments, the method further comprises providingoligonucleotides comprising a second plurality of unsaturated moietiesselected from cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants or combinationsthereof; and reacting the second plurality of unsaturated moieties ofthe oligonucleotides with the functional groups of the functionalizedmolecule to form covalent bonding.

Some embodiments described herein relate to a method of grafting primersto a first surface of a substrate, the method comprising:

providing a substrate comprising a coating layer on a first surface ofthe substrate, wherein the coating layer comprises silane or a silanederivative covalently bound to a functionalized molecule comprisingfunctional groups through a first plurality of unsaturated moieties ofthe silane or silane derivative, and wherein the first plurality ofunsaturated moieties are selected from cycloalkenes, cycloalkynes,heterocycloalkenes, heterocycloalkynes, or optionally substitutedvariants or combinations thereof;

contacting oligonucleotides comprising a second plurality of unsaturatedmoieties selected from cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants or combinationsthereof with the coating layer; and

reacting the second plurality of unsaturated moieties of theoligonucleotides with the functional groups of the functionalizedmolecule to form covalent bonding. In some embodiments, the coatinglayer is prepared by applying silane or a silane derivative comprisingthe first plurality of unsaturated moieties onto the first surface ofthe substrate, and covalently attaching the functionalized molecule tothe silane or silane derivative by reacting the functional groups of thefunctionalized molecule with the first plurality of unsaturatedmoieties.

Some embodiments described herein relate to a method of grafting primerson a first surface of a substrate, the method comprising:

providing a substrate having a first surface comprising silane or asilane derivative, wherein said silane or silane derivative comprises afirst plurality of unsaturated moieties selected from cycloalkenes,cycloalkynes, heterocycloalkenes, heterocycloalkynes, or optionallysubstituted variants or combinations thereof covalently attached theretoonto the first surface of the substrate;

providing pre-conjugated primers comprising oligonucleotides covalentlyattached to a functionalized molecule, wherein said functionalizedmolecule comprises functional groups; and

contacting the pre-conjugated primers with the silane or silanederivative such that the pre-conjugated primers are covalently attachedto the first surface of the substrate by reacting the functional groupsof the functionalized molecule with the first plurality of unsaturatedmoieties of the silane or silane derivative to form covalent bonding.

In some embodiments, the first surface of the substrate is pre-treatedwith silane or a silane derivative described herein. In someembodiments, the pre-conjugated primers are prepared by reacting asecond plurality of unsaturated moieties of the oligonucleotides withthe functional groups of the functionalized molecule to form covalentbonds, wherein the second plurality of unsaturated moieties are selectedfrom cycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes,or optionally substituted variants or combinations thereof.

In any of the embodiments described herein, the first plurality ofunsaturated moieties can be selected from norbornene, heteronorbornenes,norbornene derivatives, trans-cyclooctene, trans-cyclooctenederivatives, cyclooctyne, bicycloalkynes, or optionally substitutedvariants or combinations thereof. In some other embodiments, any othercycloalkenes, cycloalkynes, heterocycloalkenes, or heterocycloalkynespresenting ring strain can also be used. In some embodiments, the thefirst plurality of unsaturated moieties can be optionally substitutednorbornenes. In some embodiments, the first plurality of unsaturatedmoieties can be optionally substituted cyclooctyne. In some embodiment,the first plurality of unsaturated moieties can be selected fromoptionally substituted bicyclononynes. In some embodiments, theoptionally substituted bicyclononynes comprise bicyclo[6.1.0]non-4-yne.

In any of the embodiments described herein, the second plurality ofunsaturated moieties of the oligonucleotides can be selected fromnorbornene, heteronorbornenes, norbornene derivatives,trans-cyclooctene, trans-cyclooctene derivatives, cyclooctyne,bicycloalkynes, or optionally substituted variants or combinationsthereof. In some other embodiments, any other cycloalkenes,cycloalkynes, heterocycloalkenes, or heterocycloalkynes presenting ringstrain can also be used. In some embodiments, the second plurality ofunsaturated moieties can be optionally substituted cyclooctyne. In someembodiments, the second plurality of unsaturated moieties can beoptionally substituted bicyclononynes. In some further embodiments, theoptionally substituted bicyclononynes comprise bicyclo[6.1.0]non-4-yne.

In any of the embodiments described herein, the silane or silanederivative can comprise the following formula:

where R¹, R² and R³ can each be independently selected from hydrogen,halogen, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted aryl, optionally substituted aryloxy, optionallysubstituted heteroaryl or optionally substituted heteroaryloxy. In someembodiments, each R¹, R² and R³ can be independently selected fromoptionally substituted alkoxy. In some such embodiments, each of R¹, R²and R³ is methoxy.

In any of the embodiments described herein, the silane or silanederivative can be applied onto the first surface by chemical vapordeposition. In some other embodiments, the silane or silane derivativecan be applied onto the first surface by Yield Engineering Systems (YES)method.

In any of the embodiments described herein, the silane or silanederivative further comprises linkers covalently attached between siliconatoms of the silane or silane derivative and the first plurality ofunsaturated moieties. In some such embodiments, the linkers are selectedfrom optionally substituted alkylenes, optionally substitutedheteroalkylenes, optionally substituted cycloalkylenes, optionallysubstituted heterocyclylenes, optionally substituted arylenes,optionally substituted heteroarylenes, optionally substitutedpolyethylene glycols, cleavable linkers, or combinations thereof. Insome such embodiments, the linkers are optionally substituted alkylenelinkers. In some further such embodiments, the linkers are optionallysubstituted ethylene linker. In some other such embodiments, the linkersare cleavable linkers. In some such embodiments, the cleavable linkersare selected from (—S—S—), esters, nitrobenzene, imines, peptides,oligonucleotides, or polynucleotides.

In any of the embodiments described herein, the functionalized moleculecomprises a polymer, a hydrogel, an amino acid, a peptide, a nucleoside,a nucleotide, a polynucleotide, a protein, or combinations thereof. Insome embodiments, the functionalized molecule is selected from apolymer, a hydrogel, an amino acid, a peptide, a nucleoside, anucleotide, a polynucleotide, a protein, or combinations thereof. Insome further embodiments, the functionalized molecule is a hydrogel or apolymer comprising one or more functional groups selected from azido,optionally substituted amino, optionally substituted alkenyl, optionallysubstituted hydrazone, optionally substituted hydrazine, carboxyl,hydroxy, optionally substituted tetrazole, optionally substitutedtetrazine, nitrile oxide, nitrone or thiol. In some embodiments, thefunctionalized molecule comprises a polymer or hydrogel comprising arecurring unit of Formula (I) and a recurring unit of formula (II) asdescribed below in the Detailed Description of Embodiments. In someembodiments, the functionalized molecule comprises a polymer comprisingFormula (III) or (III′) as described below in the Detailed Descriptionof Embodiments.

In any of the embodiments described herein, the functionalized moleculecomprises functional groups selected from optionally substitutedalkenyl, azido, optionally substituted amino, carboxyl, optionallysubstituted hydrazone, optionally substituted hydrazine, hydroxy,optionally substituted tetrazole, optionally substituted tetrazine,nitrile oxide, nitrone, or thiol, provided that the functionalizedmolecule is not norbornene or polymerized norbornene. In some suchembodiments, the functionalized molecule comprises azido groups. In someembodiments, the functionalized molecule ispoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM).

In any of the embodiments described herein, the substrate is selectedfrom glass substrate, silica substrate, quartz, plastic substrate, metalsubstrate, metal oxide substrate, or combinations thereof. In oneembodiment, the substrate is a glass substrate.

In any of the embodiments described herein, the first surface comprisesboth functionalized molecules coated regions and inert regions. In someembodiments, the inert regions are selected from glass regions, metalregions, mask regions and interstitial regions, or combinations thereof.In one embodiment, the inert regions comprise glass.

In any of the embodiments of the methods described herein, the methodcan further comprise a washing step to remove excess unboundedfunctionalized molecules.

In any of the embodiments of the methods described herein, the methodcan further comprise a drying step.

In any of the embodiments of the methods for grafting primers asdescribed herein, the method can further comprise a washing step toremove excess unbounded oligonucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a glass substrate silanized with a norbornene-silanederivative [(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane (1a) andsubsequently coated and thermally cross-linked with PAZAM.

FIG. 1B shows the related chart of median Typhoon intensity of thegrafted norbornene silane derivative (1a) silanized/PAZAM coatedunpatterned surface hybridized with a complimentary TET dye-containingoligonucleotide sequence.

FIG. 2A shows a glass substrate patterned with nanowells silanized withthe norbornene-silane derivative (1a) and subsequently coated andthermally cross-linked with PAZAM.

FIG. 2B shows the related chart of median fluorescence intensities ofthe grafted norbornene silane derivative (1a) silanized/PAZAM coatedunpatterned surface hybridized with a complimentary TET dye-containingoligonucleotide sequence.

FIG. 3A shows a Typhoon fluorescence image of the grafted surfacehybridized with a complimentary dye-containing oligonucleotide sequenceusing acrylamide functionalized substrate.

FIG. 3B shows a Typhoon fluorescence image of the grafted surfacehybridized with a complimentary dye-containing oligonucleotide sequenceusing norbornene silane derivative (1a) silanized PAZAM coatedsubstrate.

FIG. 3C shows that a 0.25% aqueous PAZAM solution wets a norbornenesilane derivative (1a) silanized surface.

FIG. 4 shows the sequencing metrics of the grafted primer from thesubstrate prepared by the procedure described in Example 1.

FIG. 5A shows an initial Typhoon image of the grafted surface of asubstrate using a copper-free grafting method with BCN modified primers.

FIG. 5B is a line and bar chart that illustrates the initial TET QC dataafter grafting a PAZAM surface with BCN modified primers and surfaceloss percentage as measured after a thermal Stress Test.

FIG. 6A shows an initial Typhoon image of the grafted surface of asubstrate using copper-free grafting method and different concentrationsof BCN modified primers.

FIG. 6B is a line and bar chart that illustrates the initial TET QC dataafter grafting a PAZAM surface with different concentrations of BCNmodified primers and surface loss percentage as measured after a thermalStress Test.

FIG. 7A shows a fluorescence image of clusters grown from a BCN modifiedprimer grafted surface with low cluster density.

FIG. 7B shows a fluorescence image of clusters grown from a BCN modifiedprimer grafted surface with high cluster density.

FIGS. 8A and 8B shows a thumbnail image of clusters from both surfacesof a channel coated with a pre-conjugated PAZAM mixture obtained from astandard HiSeq system.

FIG. 9 shows a thumbnail image of clusters from one surface of a channelcoated with pre-conjugated PAZAM mixture obtained from a standard MiSeqsystem.

FIG. 10 shows a flow chart of growing thin polymer films from thesurface of a silicon wafer.

FIG. 11 illustrates a process of coating a flowcell surface pre-treatedwith a norbornene-derivatized silane layer.

DETAILED DESCRIPTION OF EMBODIMENTS

Embodiments of the invention relate to the conjugation of afunctionalized molecule to the surface of a substrate functionalizedwith silane or a silane derivative having optionally substitutedunsaturated moieties selected from cycloalkenes, cycloalkynes,heterocycloalkenes, or heterocycloalkynes covalently bonded to thesilicon atoms of the silane or silane derivative. In one embodiment, thefunctionalized molecule is a hydrogel, polymer or other molecule that isdesired to be attached to a substrate. In some embodiments, thefunctionalized molecule is conjugated to a surface through a norbornenederivative, such as a norbornene-derivatized silane, such as[(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. In someembodiments, the functionalized molecule is conjugated to a surfacethrough a cycloalkyne-derivatized silane, such as cyclooctyne or abicyclononyne-derivatized silane, for example, bicyclo[6.1.0]non-4-ynederivatized silane, or mixtures thereof.

Some embodiments relate to a flow cell for performingsequencing-by-synthesis reactions that includes a hydrogel, such aspoly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (“PAZAM”)conjugated to a glass substrate through a norbornene-derivatized silanelinkage, a cyclooctyne-derivatized silane linkage, or abicyclononyne-derivatized silane linkage.

Some embodiments related to a flow cell for performingsequencing-by-synthesis reactions that include oligonucleotides, such asa P5 or P7 primer conjugated to a hydrogel or polymer coated substratesurface through cyclooctyne or bicyclononyne-derivatized linkage, suchas bicyclo[6.1.0]non-4-yne derivatized linkage.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. All patents, applications, published applications and otherpublications referenced herein are incorporated by reference in theirentirety unless stated otherwise. In the event that there are aplurality of definitions for a term herein, those in this sectionprevail unless stated otherwise. As used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, biochemistry, recombinant DNA techniques andpharmacology are employed. The use of “or” or “and” means “and/or”unless stated otherwise. Furthermore, use of the term “including” aswell as other forms, such as “include”, “includes,” and “included,” isnot limiting. As used in this specification, whether in a transitionalphrase or in the body of the claim, the terms “comprise(s)” and“comprising” are to be interpreted as having an open-ended meaning. Thatis, the terms are to be interpreted synonymously with the phrases“having at least” or “including at least.” When used in the context of aprocess, the term “comprising” means that the process includes at leastthe recited steps, but may include additional steps. When used in thecontext of a compound, composition, or device, the term “comprising”means that the compound, composition, or device includes at least therecited features or components, but may also include additional featuresor components.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, common organic abbreviations are defined as follows:

-   -   Ac Acetyl    -   Ac₂O Acetic anhydride    -   APTS aminopropyl silane    -   APTES (3-aminopropyl)triethoxysilane    -   APTMS (3-aminopropyl)trimethoxysilane    -   aq. Aqueous    -   Azapa N-(5-azidoacetamidylpentyl) acrylamide    -   BCN Bicyclo[6.1.0] non-4-yne    -   BHT Butylated hydroxyl toluene    -   Bn Benzyl    -   Brapa or BRAPA N-(5-bromoacetamidylpentyl) acrylamide    -   Bz Benzoyl    -   BOC or Boc tert-Butoxycarbonyl    -   Bu n-Butyl    -   cat. Catalytic    -   Cbz Carbobenzyloxy    -   CMP Chemical mechanical polishing    -   CyCl Cyanuric chloride    -   CVD Chemical vapor deposition    -   ° C. Temperature in degrees Centigrade    -   dATP Deoxyadenosine triphosphate    -   dCTP Deoxycytidine triphosphate    -   dGTP Deoxyguanosine triphosphate    -   dTTP Deoxythymidine triphosphate    -   DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene    -   DCA Dichloroacetic acid    -   DCE 1,2-Dichloroethane    -   DCM Methylene chloride    -   DIPEA Diisopropylethylamine    -   DMA Dimethylacetamide    -   DME Dimethoxyethane    -   DMF N,N′-Dimethylformamide    -   DMSO Dimethylsulfoxide    -   DPPA Diphenylphosphoryl azide    -   Et Ethyl    -   EtOAc Ethyl acetate    -   g Gram(s)    -   GPC Gel permeation chromatography    -   h or hr Hour(s)    -   iPr Isopropyl    -   KPi 10 mM potassium phosphate buffer at pH 7.0    -   KPS Potassium persulfate    -   IPA Isopropyl Alcohol    -   LCMS Liquid chromatography-mass spectrometry    -   LDA Lithium diisopropylamide    -   m or min Minute(s)    -   mCPBA meta-Chloroperoxybenzoic Acid    -   MeOH Methanol    -   MeCN Acetonitrile    -   mL Milliliter(s)    -   MTBE Methyl tertiary-butyl ether    -   NaN₃ Sodium Azide    -   NHS N-hydroxysuccinimide    -   NHS-AA Acrylic acid N-hydroxysuccinimide ester    -   PAZAM poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide)        of any acrylamide to Azapa ratio    -   PG Protecting group    -   Ph Phenyl    -   PMDTA N,N,N′, N″, N″-Pentamethyldiethylenetriamine    -   ppt Precipitate    -   rt Room temperature    -   SBS Sequencing-by-Synthesis    -   SFA Silane Free Acrylamide as defined in U.S. Pat. Pub. No.        2011/0059865    -   Sulfo-HSAB or SHSAB N-Hydroxysulfosuccinimidyl-4-azidobenzoate    -   TEA Triethylamine    -   TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl    -   TCDI 1,1′-Thiocarbonyl diimidazole    -   Tert, t tertiary    -   TFA Trifluoroacetic acid    -   THF Tetrahydrofuran    -   TEMED Tetramethylethylenediamine    -   YES Yield Engineering Systems    -   μL Microliter(s)

As used herein, the term “array” refers to a population of differentprobe molecules that are attached to one or more substrates such thatthe different probe molecules can be differentiated from each otheraccording to relative location. An array can include different probemolecules that are each located at a different addressable location on asubstrate. Alternatively or additionally, an array can include separatesubstrates each bearing a different probe molecule, wherein thedifferent probe molecules can be identified according to the locationsof the substrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid. Exemplaryarrays in which separate substrates are located on a surface include,without limitation, those including beads in wells as described, forexample, in U.S. Pat. No. 6,355,431 B1, US 2002/0102578 and PCTPublication No. WO 00/63437. Exemplary formats that can be used in thepresent application to distinguish beads in a liquid array, for example,using a microfluidic device, such as a fluorescent activated cell sorter(FACS), are described, for example, in U.S. Pat. No. 6,524,793. Furtherexamples of arrays that can be used in the application include, withoutlimitation, those described in U.S. Pat. Nos. 5,429,807; 5,436,327;5,561,071; 5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523;6,136,269; 6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193;6,346,413; 6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO93/17126; WO 95/11995; WO 95/35505; EP 742 287; and EP 799 897.

As used herein, the term “covalently attached” or “covalently bonded”refers to the forming of a chemical bonding that is characterized by thesharing of pairs of electrons between atoms. For example, a covalentlyattached polymer coating refers to a polymer coating that forms chemicalbonds with a functionalized surface of a substrate, as compared toattachment to the surface via other means, for example, adhesion orelectrostatic interaction. It will be appreciated that polymers that areattached covalently to a surface can also be bonded via means inaddition to covalent attachment, for example, physisorption.

As used herein, “C_(a) to C_(b)” or “C_(a-b)” in which “a” and “b” areintegers refer to the number of carbon atoms in the specified group.That is, the group can contain from “a” to “b”, inclusive, carbon atoms.Thus, for example, a “C₁ to C₄ alkyl” or “C₁₋₄ alkyl” group refers toall alkyl groups having from 1 to 4 carbons, that is, CH₃—, CH₃CH₂—,CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—.

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 4 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of exampleonly, “C₁₋₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C₁₋₉ alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkylthio” refers to the formula —SR wherein R is analkyl as is defined above, such as “C₁₋₉ alkylthio” and the like,including but not limited to methylmercapto, ethylmercapto,n-propylmercapto, 1-methylethylmercapto (isopropylmercapto),n-butylmercapto, iso-butylmercapto, sec-butylmercapto,tert-butylmercapto, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₋₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include,but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, andhexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 4carbon atoms. The alkynyl group may be designated as “C₂₋₄ alkynyl” orsimilar designations. By way of example only, “C₂₋₄ alkynyl” indicatesthat there are two to four carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

As used herein, “heteroalkyl” refers to a straight or branchedhydrocarbon chain containing one or more heteroatoms, that is, anelement other than carbon, including but not limited to, nitrogen,oxygen and sulfur, in the chain backbone. The heteroalkyl group may have1 to 20 carbon atoms, although the present definition also covers theoccurrence of the term “heteroalkyl” where no numerical range isdesignated. The heteroalkyl group may also be a medium size heteroalkylhaving 1 to 9 carbon atoms. The heteroalkyl group could also be a lowerheteroalkyl having 1 to 4 carbon atoms. The heteroalkyl group may bedesignated as “C₁₋₄ heteroalkyl” or similar designations. Theheteroalkyl group may contain one or more heteroatoms. By way of exampleonly, “C₁₋₄ heteroalkyl” indicates that there are one to four carbonatoms in the heteroalkyl chain and additionally one or more heteroatomsin the backbone of the chain.

As used herein, “alkylene” means a branched, or straight chain fullysaturated di-radical chemical group containing only carbon and hydrogenthat is attached to the rest of the molecule via two points ofattachment (i.e., an alkanediyl). The alkylene group may have 1 to20,000 carbon atoms, although the present definition also covers theoccurrence of the term alkylene where no numerical range is designated.The alkylene group may also be a medium size alkylene having 1 to 9carbon atoms. The alkylene group could also be a lower alkylene having 1to 4 carbon atoms. The alkylene group may be designated as “C₁₋₄alkylene” or similar designations. By way of example only, “C₁₋₄alkylene” indicates that there are one to four carbon atoms in thealkylene chain, i.e., the alkylene chain is selected from the groupconsisting of methylene, ethylene, ethan-1,1-diyl, propylene,propan-1,1-diyl, propan-2,2-diyl, 1-methyl-ethylene, butylene,butan-1,1-diyl, butan-2,2-diyl, 2-methyl-propan-1,1-diyl,1-methyl-propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene,1,2-dimethyl-ethylene, and 1-ethyl-ethylene.

As used herein, the term “heteroalkylene” refers to an alkylene chain inwhich one or more skeletal atoms of the alkylene are selected from anatom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus orcombinations thereof. The heteroalkylene chain can have a length of 2 to20,000. Exemplary heteroalkylenes include, but are not limited to,—OCH₂—, —OCH(CH₃)—, —OC(CH₃)₂—, —OCH₂CH₂—, —CH(CH₃)O—, —CH₂OCH₂—,—CH₂OCH₂CH₂—, —SCH₂—, —SCH(CH₃)—, —SC(CH₃)₂—, —SCH₂CH₂—, —CH₂SCH₂CH₂—,—NHCH₂—, —NHCH(CH₃)—, —NHC(CH₃)₂—, —NHCH₂CH₂—, —CH₂NHCH₂—,—CH₂NHCH₂CH₂—, and the like.

As used herein, “alkenylene” means a straight or branched chaindi-radical chemical group containing only carbon and hydrogen andcontaining at least one carbon-carbon double bond that is attached tothe rest of the molecule via two points of attachment. The alkenylenegroup may have 2 to 20,000 carbon atoms, although the present definitionalso covers the occurrence of the term alkenylene where no numericalrange is designated. The alkenylene group may also be a medium sizealkenylene having 2 to 9 carbon atoms. The alkenylene group could alsobe a lower alkenylene having 2 to 4 carbon atoms. The alkenylene groupmay be designated as “C₂₋₄ alkenylene” or similar designations. By wayof example only, “C₂₋₄ alkenylene” indicates that there are two to fourcarbon atoms in the alkenylene chain, i.e., the alkenylene chain isselected from the group consisting of ethenylene, ethen-1,1-diyl,propenylene, propen-1,1-diyl, prop-2-en-1,1-diyl, 1-methyl-ethenylene,but-1-enylene, but-2-enylene, but-1,3-dienylene, buten-1,1-diyl,but-1,3-dien-1,1-diyl, but-2-en-1,1-diyl, but-3-en-1,1-diyl,1-methyl-prop-2-en-1,1-diyl, 2-methyl-prop-2-en-1,1-diyl,1-ethyl-ethenylene, 1,2-dimethyl-ethenylene, 1-methyl-propenylene,2-methyl-propenylene, 3-methyl-propenylene, 2-methyl-propen-1,1-diyl,and 2,2-dimethyl-ethen-1,1-diyl.

As used herein, “alkynylene” means a straight or branched chaindi-radical chemical group containing only carbon and hydrogen andcontaining at least one carbon-carbon triple bond that is attached tothe rest of the molecule via two points of attachment.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e.,two or more fused rings that share two adjacent carbon atoms) containingonly carbon in the ring backbone. When the aryl is a ring system, everyring in the system is aromatic. The aryl group may have 6 to 18 carbonatoms, although the present definition also covers the occurrence of theterm “aryl” where no numerical range is designated. In some embodiments,the aryl group has 6 to 10 carbon atoms. The aryl group may bedesignated as “C₆₋₁₀ aryl,” “C₆ or C₁₀ aryl,” or similar designations.Examples of aryl groups include, but are not limited to, phenyl,naphthyl, azulenyl, and anthracenyl.

As used herein, “arylene” refers to an aromatic ring or ring systemcontaining only carbon and hydrogen that is attached to the rest of themolecule via two points of attachment.

As used herein, “aryloxy” and “arylthio” refers to RO— and RS—, in whichR is an aryl as is defined above, such as “C₆₋₁₀ aryloxy” or “C₆₋₁₀arylthio” and the like, including but not limited to phenyloxy.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₄ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

As used herein, “heteroarylene” refers to an aromatic ring or ringsystem containing one or more heteroatoms in the ring backbone that isattached to the rest of the molecule via two points of attachment.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁₋₄alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃₋₆carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

A “(carbocyclyl)alkyl” is a carbocyclyl group connected, as asubstituent, via an alkylene group, such as “C₄₋₁₀ (carbocyclyl)alkyl”and the like, including but not limited to, cyclopropylmethyl,cyclobutylmethyl, cyclopropylethyl, cyclopropylbutyl, cyclobutylethyl,cyclopropylisopropyl, cyclopentylmethyl, cyclopentylethyl,cyclohexylmethyl, cyclohexylethyl, cycloheptylmethyl, and the like. Insome cases, the alkylene group is a lower alkylene group.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkylene” means a fully saturated carbocyclyl ringor ring system that is attached to the rest of the molecule via twopoints of attachment.

As used herein, “cycloalkenyl” or “cycloalkene” means a carbocyclyl ringor ring system having at least one double bond, wherein no ring in thering system is aromatic. An example is cyclohexenyl or cyclohexene.Another example is norbornene or norbornenyl.

As used herein, “heterocycloalkenyl” or “heterocycloalkene” means acarbocyclyl ring or ring system with at least one heteroatom in ringbackbone, having at least one double bond, wherein no ring in the ringsystem is aromatic.

As used herein, “cycloalkynyl” or “cycloalkyne” means a carbocyclyl ringor ring system having at least one triple bond, wherein no ring in thering system is aromatic. An example is cyclooctyne. Another example isbicyclononyne.

As used herein, “heterocycloalkynyl” or “heterocycloalkyne” means acarbocyclyl ring or ring system with at least one heteroatom in ringbackbone, having at least one triple bond, wherein no ring in the ringsystem is aromatic.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O, N or S, and inpreferred five membered monocyclic heterocyclyls, the heteroatom(s) areselected from one or two heteroatoms selected from O, N, or S. Examplesof heterocyclyl rings include, but are not limited to, azepinyl,acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

As used herein, “heterocyclylene” means a non-aromatic cyclic ring orring system containing at least one heteroatom that is attached to therest of the molecule via two points of attachment.

A “(heterocyclyl)alkyl” is a heterocyclyl group connected, as asubstituent, via an alkylene group. Examples include, but are notlimited to, imidazolinylmethyl and indolinylethyl.

As used herein, “acyl” refers to —C(═O)R, wherein R is hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, andacryl.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

An “acetal” group refers to RC(H)(OR′)₂, in which R and R′ areindependently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, and5-10 membered heterocyclyl, as defined herein.

A “cyano” group refers to a “—CN” group.

A “cyanato” group refers to an “—OCN” group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—SCN” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “sulfinyl” group refers to an “—S(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “nitrile oxide” as used herein, refers to a “RC≡N⁺O⁻” group in whichR is selected from hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, or 5-10 memberedheterocyclyl, as defined herein. Non-limiting examples of preparingnitrile oxide include in situ generation from aldoximes by treatmentwith chloramide-T or through action of base on imidoyl chlorides[RC(Cl)═NOH].

An “nitrone” as used herein, refers to a “R_(A)R_(B)C═NR_(c) ⁺O⁻” groupin which R_(A), R_(B) and R_(c) are each independently selected fromhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, asdefined herein.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-carbamyl” group refers to an “—N(R_(A))OC(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-thiocarbamyl” group refers to a “—OC(═S)NR_(A)R_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

An “N-thiocarbamyl” group refers to an “—N(R_(A))OC(═S)R_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

An “aminoalkyl” group refers to an amino group connected via an alkylenegroup.

An “alkoxyalkyl” group refers to an alkoxy group connected via analkylene group, such as a “C₂₋₈ alkoxyalkyl” and the like.

The term “hydrazine” or “hydrazinyl” as used herein refers to a —NHNH₂group.

The term “hydrazone” or “hydrazonyl” as used herein refers to a

group.

The term “formyl” as used herein refers to a —C(O)H group.

The term “epoxy” as used herein refers to

The term “ester” as used herein refers to R—C(═O)O—R′, wherein R and R′can be independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,(heteroalicyclyl)alkyl, or optionally substituted variants thereof.

The term “carboxylic acid” or “carboxyl” as used herein refers to—C(O)OH.

The term “thiocyanate” as used herein refers to —S—C≡N group.

The term “oxo-amine” as used herein refers to —O—NH₂ group, wherein oneor more hydrogen of the —NH₂ can be optionally substituted by a R group.R can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,or (heteroalicyclyl)alkyl.

As used herein, a “nucleotide” includes a nitrogen containingheterocyclic base, a sugar, and one or more phosphate groups. They aremonomeric units of a nucleic acid sequence. In RNA, the sugar is aribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl groupthat is present at the 2′ position in ribose. The nitrogen containingheterocyclic base can be purine or pyrimidine base. Purine bases includeadenine (A) and guanine (G), and modified derivatives or analogsthereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil(U), and modified derivatives or analogs thereof. The C-1 atom ofdeoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine.

As used herein, a “nucleoside” is structurally similar to a nucleotide,but lacks any phosphate moieties at the 5′ position. The term“nucleoside” is used herein in its ordinary sense as understood by thoseskilled in the art. Examples include, but are not limited to, aribonucleoside comprising a ribose moiety and a deoxyribonucleosidecomprising a deoxyribose moiety. A modified pentose moiety is a pentosemoiety in which an oxygen atom has been replaced with a carbon and/or acarbon has been replaced with a sulfur or an oxygen atom. A “nucleoside”is a monomer that can have a substituted base and/or sugar moiety.Additionally, a nucleoside can be incorporated into larger DNA and/orRNA polymers and oligomers.

As used herein, the term “polynucleotide” refers to nucleic acids ingeneral, including DNA (e.g. genomic DNA cDNA), RNA (e.g. mRNA),synthetic oligonucleotides and synthetic nucleic acid analogs.Polynucleotides may include natural or non-natural bases, orcombinations thereof and natural or non-natural backbone linkages, e.g.phosphorothioates, PNA or 2′-O-methyl-RNA, or combinations thereof.

As used herein, a “BCN primer” or “BCN modified primer” refers to aprimer comprising covalently attached bicyclo[6.1.0] non-4-yne at the 5′terminus. The primer is defined as a single strand DNA (ssDNA) moleculewith a free 3′ OH group and a modification at the 5′ terminus to allowthe coupling reactions. The primer length can be any number of baseslong and can include a variety of non natural nucleotides.

As used herein, the term “silane” refers to an organic or inorganiccompound containing one or more silicon atoms. Non-limiting example ofan inorganic silane compound is SiH₄, or halogenated SiH₄ where hydrogenis replaced by one or more halogen atoms. Non-limiting example of anorganic silane compound is X—R^(C)—Si(OR^(D))₃, wherein X is anon-hydrolyzable organic group, such as amino, vinyl, epoxy,methacrylate, sulfur, alkyl, alkenyl, alkynyl; R^(C) is a spacer, forexample —(CH₂)_(n)—, wherein n is 0 to 1000; R^(D) is selected fromhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted 5-10 memberedheteroaryl, and optionally substituted 5-10 membered heterocyclyl, asdefined herein. As used herein, the term “silane” can comprise mixturesof different silane compounds.

As used herein, the term “tetrazine” or “tetrazinyl” refers tosix-membered heteroaryl group comprising four nitrogen atoms. Tetrazinecan be optionally substituted.

As used herein, the term “tetrazole” or “tetrazolyl” refers to fivemembered heterocyclic group comprising four nitrogen atoms. Tetrazolecan be optionally substituted.

As used herein, the term “unsaturated moiety” refers to a chemical groupincludes cycloalkenes, cycloalkynes, heterocycloalkenes,heterocycloalkynes, or optionally substituted variants thereofcomprising at least one double bond or one triple bond. The unsaturatedmoieties can be mono-valent or di-valent. When the unsaturated moiety ismono-valent, cycloalkene, cycloalkyne, heterocycloalkene,heterocycloalkyne are used interchangeably with cycloalkenyls,cycloalkynyls, heterocycloalkenyl, heterocycloalkynyl. When theunsaturated moiety is di-valent, cycloalkene, cycloalkyne,heterocycloalkene, heterocycloalkyne are used interchangeably withcycloalkenylene, cycloalkynylene, heterocycloalkenylene,heterocycloalkynylene.

As used herein, the term “polymer” refers to a molecule composed of manyrepeated subunits. Polymers can be linear, branched, or hyperbranched.Non-limiting examples of branched polymers include star polymers, combpolymers, brush polymers, dendronized polymers, ladders, and dendrimers.The polymers described herein can also be in the form of polymernanoparticles.

As used herein, the prefixes “photo” or “photo-” mean relating to lightor electromagnetic radiation. The term can encompass all or part of theelectromagnetic spectrum including, but not limited to, one or more ofthe ranges commonly known as the radio, microwave, infrared, visible,ultraviolet, X-ray or gamma ray parts of the spectrum. The part of thespectrum can be one that is blocked by a metal region of a surface suchas those metals set forth herein. Alternatively or additionally, thepart of the spectrum can be one that passes through an interstitialregion of a surface such as a region made of glass, plastic, silica, orother material set forth herein. In particular embodiments, radiationcan be used that is capable of passing through a metal. Alternatively oradditionally, radiation can be used that is masked by glass, plastic,silica, or other material set forth herein.

As used herein, the term “reactive site” means a site on thefunctionalized molecule coatings described herein that can be used toattach one or more molecules by way of a chemical reaction or molecularinteraction. Such attachment may be via a covalent bond or through otherbonding or interactive forces.

As used herein, the term “YES method” refers to the chemical vapordeposition tool provided by Yield Engineering Systems (“YES”) withchemical vapor deposition process developed by Illumina, Inc. It includethree different vapor deposition systems. The automated YES-VertaCoatsilane vapor system is designed for volume production with a flexiblewafer handling module that can accommodate 200 or 300 mm wafers. Themanual load YES-1224P Silane Vapor System is designed for versatilevolume production with its configurable large capacity chambers.Yes-LabKote is a low-cost, tabletop version that is ideal forfeasibility studies and for R&D.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substituents independently selected fromC₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇carbocyclyl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heterocyclyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheterocyclyl-C₁-C₆-alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), aryl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heteroaryl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10 memberedheteroaryl(C₁-C₆)alkyl (optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo, cyano,hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether), aryloxy,sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃), halo(C₁-C₆)alkoxy(e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino, amino(C₁-C₆)alkyl,nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido,N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl,cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl,and oxo (═O). Wherever a group is described as “optionally substituted”that group can be substituted with the above substituents.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated. Thus, for example, a substituent depicted as -AE-or

includes the substituent being oriented such that the A is attached atthe leftmost attachment point of the molecule as well as the case inwhich A is attached at the rightmost attachment point of the molecule.

Where the compounds disclosed herein have at least one stereocenter,they may exist as individual enantiomers and diastereomers or asmixtures of such isomers, including racemates. Separation of theindividual isomers or selective synthesis of the individual isomers isaccomplished by application of various methods which are well known topractitioners in the art. Unless otherwise indicated, all such isomersand mixtures thereof are included in the scope of the compoundsdisclosed herein. Furthermore, compounds disclosed herein may exist inone or more crystalline or amorphous forms. Unless otherwise indicated,all such forms are included in the scope of the compounds disclosedherein including any polymorphic forms. In addition, some of thecompounds disclosed herein may form solvates with water (i.e., hydrates)or common organic solvents. Unless otherwise indicated, such solvatesare included in the scope of the compounds disclosed herein.

As used herein, the term “percent surface remaining” can refer to theintensity measured using a TET QC to stain the P5/P7 surface primers.The P5 and P7 primers are used on the surface of commercial flow cellssold by Illumina Inc. for sequencing on the HiSeq, MiSeq, GenomeAnalyzer and NextSeq platforms. The primer sequences are described inU.S. Pat. Pub. No. 2011/0059865 A1, which is incorporated herein byreference. TET is a dye labeled oligonucleotide having complimentarysequence to the P5/P7 primers. TET can be hybridized to the P5/P7primers on a surface; the excess TET can be washed away, and theattached dye concentration can be measured by fluorescence detectionusing a scanning instrument such as a Typhoon Scanner (GeneralElectric).

The skilled artisan will recognize that some structures described hereinmay be resonance forms or tautomers of compounds that may be fairlyrepresented by other chemical structures; the artisan recognizes thatsuch structures may only represent a very small portion of a sample ofsuch compound(s). Such compounds are considered within the scope of thestructures depicted, though such resonance forms or tautomers are notrepresented herein.

Silane or Silane Derivatives

Some embodiments disclosed herein relate to silane or silane derivativescomprising a plurality of unsaturated moieties selected fromcycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes oroptionally substituted variants or combinations thereof. As used herein,“cycloalkene” means a carbocyclyl ring or ring system having at leastone double bond, wherein no ring in the ring system is aromatic. As usedherein, “heterocycloalkene” means a carbocyclyl ring or ring systemcontains at least one heteroatom in ring backbone, having at least onedouble bond, wherein no ring in the ring system is aromatic. As usedherein, “cycloalkyne” means a carbocyclyl ring or ring system having atleast one triple bond, wherein no ring in the ring system is aromatic.As used herein, “heterocycloalkyne” means a carbocyclyl ring or ringsystem contains at least one heteroatom in ring backbone, having atleast one triple bond, wherein no ring in the ring system is aromatic.In some embodiments, the heteroatom in the heterocycloalkene is selectedfrom the group consisting of N, O or S. Both cycloalkene andheterocycloalkene can be optionally substituted. The unsaturatedmoieties can be mono-valent or di-valent. The unsaturated moieties canbe covalently attached either directly to the silicon atoms of thesilane or silane derivatives, or indirectly attached via linkers. Theunsaturated moieties can be further bounded to a functionalizedmolecule. In some embodiments, the unsaturated moieties are optionallysubstituted cycloalkenes, such as norbornene and derivatives thereof. Insome embodiments, the unsaturated moieties are optionally substitutedcyclooctyne or bicyclononynes. Other cycloalkenes, heterocycloalkenes,cycloalkynes, heterocycloalkynes presenting ring strain can also be usedas unsaturated moieties.

Norbornenes

In some embodiments, the cycloalkene is norbornene or a norbornenederivative.

In some embodiments, norbornene can be substituted with one or moresubstituents selected from selected from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇ carbocyclyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), 5-10 membered heterocyclyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),aryl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O).

Alternatively, the two adjacent substituents on norbornene can formadditional rings. For example,

represents a di-substituted norbornene, wherein R^(a) and R^(b),together with the atom to which they are attached, can be joinedtogether to form an optionally substituted aryl, an optionallysubstituted heteroaryl, an optionally substituted cycloalkyl or anoptionally substituted heterocyclyl.

In some embodiments, norbornene can be replaced by other cycloalkenes.No limiting examples include optionally substituted trans-cyclooctene,optionally substituted trans-cyclopentene, optionally substitutedtrans-cycloheptene, optionally substituted trans-cyclononene, optionallysubstituted bicyclo[3.3.1]non-1-ene, optionally substitutedbicyclo[4.3.1]dec-1(9)-ene, optionally substitutedbicyclo[4.2.1]non-1(8)-ene, and optionally substitutedbicyclo[4.2.1]-non-1-ene.

Hetero(norbornenes)

In some embodiments, the heterocycloalkene used herein is aheteronorbornene. As used herein, (hetero)norbornene means one or morecarbon atoms in a norbornene molecule in replaced by one or moreheteroatoms. Non-limiting examples of (hetero)norbornene include

or optionally substituted variants thereof.

Exemplary Norbornene Reactions

A. 1,3-Dipolar Cycloaddition with Azides

The reaction of organic azides and olefinic bonds leading to theformation of 1,2,3-triazoline was first reported by Wolff in 1912. SeeWolff, Liebigs. Ann., 1912, 394, 23. This type of reaction is termed as1,3-dipolar cycloaddition. Azide additions to terminal alkyne wererecognized as an example of 1,3-dipolar cycloaddition reaction from theresearch of Huisgen. See Proceedings of the Chemical Society, 1961,357-396. Scheiner et al. reported the kinetic investigation of arylazides to norbornene (see Scheiner et al., J. Am. Chem. Soc, 1965, 87,306-311). The general reaction scheme is shown as follows:

In addition, Shea et al. reported studies of reactivity of torsionallystrained double bonds in 1,3-dipolar cycloadditions with2,4,6-trinitrophenyl azide. A series of mono- and bi-cyclic olefinsincluding trans-cycloalkenes and bridgehead alkenes were tested. See,Shea et al., J. Am. Chem. Soc. 1992, 114, 4846-4855.

B. Coupling Reaction with Tetrazines

The additive-free “click” reaction for polymer functionalization andcoupling by the inverse electron demand Diels-Alder (DA_(inv)) reactionof tetrazine and norbornene was reported by Hansell et al. (see Hansellet al., J. Am. Chem. Soc. 2011, 133, 13828-13831). The general reactionscheme is shown as follows:

Other exemplary metal-free click reactions includes the(hetero-)Diels-Alder and the radical based thiol-ene reaction, asreported by Hoyle et al., Chem. Soc. Rev. 2010, 39, 1355-1387.

C. Coupling Reaction with Tetrazoles and Hydrazones

Kaya et al. reported a norbornene amino acid (1) for proteinmodification in a copper-free click reaction. See Kaya et al., Angew.Chem. Int. Ed. 2012, 51, 4466-4469. In the first example, a nitrileimine was generated by base-promoted HCl elimination from the hydrazonylchloride and then used in a cycloaddition reaction with the norbornenederivative (1). In the second example, the nitrile imine was generatedfrom a tetrazole in a photo-chemical reaction.

D. Ring-Opening Reactions with Olefins

Kim et al. reported a method for growing thin polymer films from thesurface of a silicon wafer bearing a native oxide (Si/SiO₂) by using asurface-initiated ring-opening metathesis polymerization of norbornene.FIG. 10 outlines a three-step procedure: (i) the formation of aself-assembled monolayer on silicon that comprising norbornenyl groups;(ii) the attachment of a ruthenium catalyst [(Cy₃P)₂Cl₂Ru═CHPh,Cy=cyclohexyl] to the surface; and (iii) the polymerization of addedmonomers to generate the film. See Kim et al., Macromolecules 2000, 33,2793-2795.

Similarly, Liu et al. reported the region-selectivering-opening/cross-metathesis reactions of norbornene derivatives withelectron-rich olefins, catalyzed by a ruthenium catalyst, as shown inthe scheme below. See Liu et al., Org. Lett. 2005, 7, 131-133.

E. Cycloaddition with Nitrile Oxides

Gutsmiedl et al. reported the first examples of strain-promoted nitrileoxide cycloaddition involved norbornene-modified DNA substrate. Thestrained alkene is suited to cycloaddition with a variety of nitrileoxides generated in situ either from hydroxamoyl chlorides or directlyby treatment of parent oxime with N-Chlorosuccinimide. See Gutsmiedl etal., Org. Lett. 2009, 11, 2405-2408.

Cyclooctyne

In some embodiments, the cycloalkyne is cyclooctyne or a cyclooctynederivative.

In some embodiments, cycloalkyne can be substituted with one or moresubstituents selected from selected from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇ carbocyclyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), 5-10 membered heterocyclyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),aryl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O).

Alternatively, the two adjacent substituents on cyclooctyne can formadditional rings. For example,

represents a di-substituted cyclooctyne, wherein R^(a) and R^(b),together with the atom to which they are attached, can be joinedtogether to form an optionally substituted aryl, an optionallysubstituted heteroaryl, an optionally substituted cycloalkyl or anoptionally substituted heterocyclyl. In some embodiments, thecyclooctyne derivative can comprise the following structures:

Strain-Promoted Azide-Alkyne Cycloaddition or Nitrile Oxide-AlkyneCycloaddition

Cyclooctynes can undergo 1,3-cycloaddition with azides. This type ofstrain-promoted azide-alkyne cycloaddition (SPAAC) reaction has beenused in the copper-free DNA ligation. Van Geel et al., has reported thata variety of strained octynes can be used to efficiently label azidetagged proteins. See R. van Geel et al., “Preventing Thiol-yne AdditionImproves the Specificity of Strain-Promoted Azide-Alkyne Cycloaddition,”Bioconjugate Chem., 2012, 23, 392-398. Similar work has also beenreported by Yao et al. who has demonstrated that strained alkynes can beused to attach fluorophores to amino acid sequences. See Tao et al,“Fluorophore targeting to cellular proteins via enzyme-mediated azideligation and strain-promoted cycloaddition,” J. Am. Chem. Soc., 2012,134, 3720-3728.

Recent studies on relative activity of nitrile oxides, nitrones asalternative to azide dipoles in reaction with cyclooctynes suggestedsuperior reactivities for strain-promoted alkyne/nitrile oxidecycloaddition. See Sanders et al., J. Am. Soc. Chem. 2011, 133, 949-957;Jawalekar et al., Chem. Commun. 2011, 47, 3198-3200; and McKay et al.,Chem. Commun. 2010, 46, 931-933. It was observed that the rate constantfor [3+2] addition of bicyclo[6.1.0]nonyne (BCN) to benzonitrile oxidewas greater by a factor of 10 than that overserved for the correspondingreaction with benzyl azide. Similar results were also observed withcycloaddition between dibenzocycloactynol (DIBO) and benzonitrile oxide(generated in situ from hydroxamoyl chloride) or benzyl azide. The studysuggests the former reaction was about 60 times faster than the latter.

Bicyclononynes

In some embodiments, the cycloalkynes can comprise bicyclic ring system,for example, bicyclononynes. In some embodiments, the bicyclononynes canbe selected from bicyclo[6.1.0]non-4-yne or derivatives thereof. In someother embodiments, the bicyclononynes can also be selected frombicyclo[6.1.0]non-2-yne or bicyclo[6.1.0]non-3-yne.

In some embodiments, bicyclononynes can be substituted with one or moresubstituents selected from selected from C₁-C₆ alkyl, C₁-C₆ alkenyl,C₁-C₆ alkynyl, C₁-C₆ heteroalkyl, C₃-C₇ carbocyclyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), 5-10 membered heterocyclyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),aryl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O).

Alternatively, the two adjacent substituents on a bicyclononyne can formadditional rings. For example,

represents a di-substituted bicyclo[6.1.0]non-4-yne, wherein R^(a) andR^(b), together with the atom to which they are attached, can be joinedtogether to form an optionally substituted aryl, an optionallysubstituted heteroaryl, an optionally substituted cycloalkyl or anoptionally substituted heterocyclyl. Bicyclononynes can undergo similarSPAAC alkyne cycloaddition with azides or nitrile oxides as describedabove with respect to cyclooctyne due to the strain in the bicyclic ringsystem.Hydrogels

Some embodiments described herein include immobilizing a functionalizedhydrogel to a surface of a substrate via unsaturated moieties of thefunctionalized silane or silane derivatives. Non-limiting examples ofhydrogels can be used in the present application are described herein.

WO 00/31148 discloses polyacrylamide hydrogels and polyacrylamidehydrogel-based arrays in which a so-called polyacrylamide prepolymer isformed, preferably from acrylamide and an acrylic acid or an acrylicacid derivative containing a vinyl group. Crosslinking of the prepolymermay then be affected. The hydrogels so produced are solid-supported,preferably on glass. Functionalization of the solid-supported hydrogelmay also be effected.

WO 01/01143 describes technology similar to WO00/31148 but differing inthat the hydrogel bears functionality capable of participating in a[2+2] photocycloaddition reaction with a biomolecule so as to formimmobilized arrays of such biomolecules. Dimethylmaleimide (DMI) is aparticularly preferred functionality. The use of [2+2]photocycloaddition reactions, in the context of polyacrylamide-basedmicroarray technology is also described in WO02/12566 and WO03/014392.

U.S. Pat. No. 6,465,178 discloses the use of reagent compositions inproviding activated slides for use in preparing microarrays of nucleicacids; the reagent compositions include acrylamide copolymers. Theactivated slides are stated to be particularly well suited to replaceconventional (e.g. silylated) glass slides in the preparation ofmicroarrays.

WO 00/53812 discloses the preparation of polyacrylamide-based hydrogelarrays of DNA and the use of these arrays in replica amplification.

Once hydrogels have been formed, molecules may then be attached to themso as to produce molecular arrays, if desired. Attachment has beeneffected in different ways in the prior art. For example, U.S. Pat. No.6,372,813 teaches immobilization of polynucleotides bearingdimethylmaleimide groups to the hydrogels produced which beardimethylmaleimide groups by conducting a [2+2] photocycloaddition stepbetween two dimethylmaleimide groups—one attached to the polynucleotideto be immobilized and one pendant from the hydrogel.

Where the molecular array is formed after generation of the hydrogel,two strategies have been employed to achieve this end. Firstly, thehydrogel may be modified chemically after it is produced. Problems withthis approach include an overall low efficiency in the preparation ofthe array and the low stability relating to the attachment chemistry,particularly upon exposure to high temperatures, ionic solutions andmultiple wash steps.

A more common alternative is to effect polymerization with a co-monomerhaving a functionality primed or pre-activated to react with themolecules to be arrayed.

Alternatives to initial formation of hydrogels followed by subsequentarraying of molecules thereto have been described in the prior art wherethe array is formed at the same time as the hydrogel is produced. Thismay be effected by, for example, direct copolymerization ofacrylamide-derivatized polynucleotides. An example of this approach isdescribed in WO01/62982 in which acrylamide-derivatized polynucleotidesare mixed with solutions, of acrylamide and polymerization is effecteddirectly.

Mosaic Technologies (Boston, Mass., USA) produce ACRYDITE™ (anacrylamide phosphoramidite) which can be reacted with polynucleotidesprior to copolymerization of the resultant monomer with acrylamide.

Efimov et al. (Nucleic Acids Research, 1999, 27 (22), 4416-4426)disclose a further example of a simultaneous formation of hydrogel/arrayin which copolymerization of acrylamide, reactive acrylic acidderivatives and the modified polynucleotides having 5′- or 3′-terminalacrylamide groups is affected.

Polymers

Some embodiments described herein include immobilizing a functionalizedpolymer to a surface of a substrate via cycloalkene or heterocycloalkenefunctionalized silane or silane derivatives. No-limiting examples of thepolymers that can be used in the present application are described inU.S. Ser. No. 13/784,368 and U.S. Pat. Pub. No. 2011/0059865, which arehereby incorporated by references in their entireties.

In some embodiments, the polymer used herein comprises a recurring unitof Formula (I) and a recurring unit of Formula (II):

wherein: R¹ is H or alkyl; R^(A) is selected from the group consistingof azido, optionally substituted amino, optionally substituted alkenyl,optionally substituted hydrazone, optionally substituted hydrazine,carboxyl, hydroxy, optionally substituted tetrazole, optionallysubstituted tetrazine, and thiol; X is an optionally substitutedalkylene linker or an optionally substituted heteroalkylene linker; R⁴,R^(4′), R⁵ and R^(5′) are each independently selected from H, R⁶, OR⁶,—C(O)OR⁶, —C(O)R⁶, —OC(O)R⁶, —C(O) NR⁷R⁸, or —NR⁷R⁸; R⁶ is independentlyselected from H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl, heteroaryl,heterocyclyl, or optionally substituted variants thereof; R⁷ and R⁸ areeach independently selected from H or alkyl, or R⁷ and R⁸ are joinedtogether with the atom or atoms to which they are attached to form aheterocycle.

In some embodiments, R^(A) is azido. In some embodiments, X is anoptionally substituted alkylene linker. In some embodiments, R¹ ishydrogen, In some other embodiments, R¹ is methyl. In some embodiments,R⁴ is hydrogen and R^(4′) is —C(O)NR⁷R⁸. In some embodiments, each of R⁵and R^(5′) is hydrogen. In some embodiments, R⁵ is hydrogen and R^(5′)is methyl.

In some embodiment, the polymer used herein comprises a polymer ofFormula (III) or (III′):

wherein R¹ is selected from H or optionally substituted alkyl; R^(A) isselected from the group consisting of azido, optionally substitutedamino, optionally substituted alkenyl, optionally substituted hydrazone,optionally substituted hydrazine, carboxyl, hydroxy, optionallysubstituted tetrazole, optionally substituted tetrazine, and thiol; eachof the —(CH₂)-p can be optionally substituted; p is an integer in therange of 1-50; R⁵ is selected from H or optionally substituted alkyl; nis an integer in the range of 1 to 50,000; and m is an integer in therange of 1 to 100,000. In some embodiments, p is 5. In some embodiments,R^(A) is azido.

PAZAM

In one embodiment, the polymer of Formula (III) or (III′) is alsorepresented by Formula (IIIa) or (IIIb):

wherein n is an integer in the range of 1-20,000, and m is an integer inthe range of 1-100,000.

In some embodiments, the functionalized molecule used for directconjugation is poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide)(PAZAM). In some embodiments, PAZAM is a linear polymer. In some otherembodiments, PAZAM is a lightly cross-linked polymer. In someembodiments, PAZAM is applied to the surface as an aqueous solution. Insome other embodiments, PAZAM is applied to the surface as an aqueoussolution with one or more solvent additives, such as ethanol. The methodfor preparation different PAZAM polymers is discussed in detail in U.S.Ser. No. 13/784,368, which is hereby incorporated by reference in itsentirety.

Substrates

In some embodiments, substrates used in the present application includesilica-based substrates, such as glass, fused silica and othersilica-containing materials. In some embodiments, silica-basedsubstrates can also be silicon, silicon dioxide, silicon nitride,silicone hydrides. In some embodiments, substrates used in the presentapplication include plastic materials such as polyethylene, polystyrene,poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonatesand poly(methyl methacrylate). Preferred plastics material arepoly(methyl methacrylate), polystyrene and cyclic olefin polymersubstrates. In some embodiments, the substrate is a silica-basedmaterial or plastic material. In one embodiment, the substrate has atleast one surface comprising glass.

In some other embodiments, the substrates can be a metal. In some suchembodiments, the metal is gold. In some embodiments, the substrate hasat least one surface comprising a metal oxide. In one embodiment, thesurface comprises a tantalum oxide.

Acrylamide, enone, or acrylate may also be utilized as a substratematerial. Other substrate materials can include, but are not limited togallium arsenide, indium phosphide, aluminum, ceramics, polyimide,quartz, resins, polymers and copolymers. The foregoing lists areintended to be illustrative of, but not limited to the presentapplication.

In some embodiments, the substrate and/or the substrate surface can bequartz. In some other embodiments, the substrate and/or the substratesurface can be semiconductor, i.e. GaAs or ITO.

Substrates can comprise a single material or a plurality of differentmaterials. Substrates can be composites or laminates. Substrate can beflat, round, textured and patterned. Patterns can be formed, forexample, by metal pads that form features on non-metallic surfaces, forexample, as described in U.S. patent application Ser. No. 13/661,524,which is incorporated herein by reference. Another useful patternedsurface is one having well features formed on a surface, for example, asdescribed in U.S. Ser. No. 13/787,396, US Pat. App. Pub. No.2011/0172118 A1 or U.S. Pat. No. 7,622,294, each of which isincorporated herein by reference. For embodiments that use a patternedsubstrate, a gel can be selectively attached to the pattern features(e.g. gel can be attached to metal pads or gel can be attached to theinterior of wells) or alternatively the gel can be uniformly attachedacross both the pattern features and the interstitial regions.

Advantages in using plastics-based substrates in the preparation and useof molecular arrays include cost: the preparation of appropriateplastics-based substrates by, for example injection-molding, isgenerally cheaper than the preparation, e.g. by etching and bonding, ofsilica-based substrates. Another advantage is the nearly limitlessvariety of plastics allowing fine-tuning of the optical properties ofthe support to suit the application for which it is intended or to whichit may be put.

Where metals are used as substrates or as pads on a substrate, this maybe because of the desired application: the conductivity of metals canallow modulation of the electric field in DNA-based sensors. In thisway, DNA mismatch discrimination may be enhanced, the orientation ofimmobilized oligonucleotide molecules can be affected, or DNAhybridization kinetics can be accelerated.

Preferably the substrate is silica-based but the shape of the substrateemployed may be varied in accordance with the application for which thepresent application is practiced. Generally, however, slides of supportmaterial, such as silica, e.g. fused silica, are of particular utilityin the preparation and subsequent integration of molecules. Ofparticular use in the practice of the present application are fusedsilica slides sold under the trade name SPECTRASIL™. Thisnotwithstanding, it will be evident to the skilled person that thepresent application is equally applicable to other presentations ofsubstrate (including silica-based supports), such as beads, rods and thelike.

In some embodiments, the surface of the substrate comprises bothfunctional molecules-coated regions and inert regions with no coatings.In some such embodiments, the functionalized molecule coatings arehydrogel or polymer coatings. The functional molecules-coated regionscan comprise reactive sites, and thus, can be used to attach moleculesthrough chemical bonding or other molecular interactions. In someembodiments, the functional molecules-coated regions (e.g. reactivefeatures, pads, beads or wells) and the inert regions (referred to asinterstitial regions) can alternate so as to form a pattern or a grid.Such patterns can be in one or two dimensions. In some embodiments, theinert regions can be selected from glass regions, metal regions, maskregions or interstitial regions, or combinations thereof. Alternativelythese materials can form reactive regions. Inertness or reactivity willdepend on the chemistry and processes used. on the substrate. In oneembodiment, the surface comprises glass regions. In another embodiment,the surface comprises metal regions. In still another embodiment, thesurface comprises mask regions. In some embodiments of the compositionsdescribed herein, the substrate can be a bead. Non-limiting exemplarysubstrate materials that can be coated with a polymer of the presentdisclosure or that can otherwise be used in a composition or method setforth herein are described in U.S. Ser. Nos. 13/492,661 and 13/661,524,each of which is incorporated herein by reference.

In some embodiments, a substrate described herein is forms at least partof a flow cell or is located in a flow cell. In some such embodiments,the flow cells further comprise polynucleotides attached to the surfaceof the substrate via the functional molecules coating, for example, apolymer coating. In some embodiments, the polynucleotides are present inthe flow cells in polynucleotide clusters, wherein the polynucleotidesof the polynucleotide clusters are attached to a surface of the flowcell via the polymer coating. In such embodiments, the surface of theflow cell body to which the polynucleotides are attached is consideredthe substrate. In other embodiments, a separate substrate having apolymer coated surface is inserted into the body of the flow cell. Inpreferred embodiments, the flow cell is a flow chamber that is dividedinto a plurality of lanes or a plurality of sectors, wherein one or moreof the plurality of lanes or plurality of sectors comprises a surfacethat is coated with a covalently attached polymer coating describedherein. In some embodiments of the flow cells described herein, theattached polynucleotides within a single polynucleotide cluster have thesame or similar nucleotide sequence. In some embodiments of the flowcells described herein, the attached polynucleotides of differentpolynucleotide clusters have different or nonsimilar nucleotidesequences. Exemplary flow cells and substrates for manufacture of flowcells that can be used in method or composition set forth hereininclude, but are not limited to, those commercially available fromIllumina, Inc. (San Diego, Calif.) or described in US 2010/0111768 A1 orUS 2012/0270305, each of which is incorporated herein by reference.

Silica-Based Substrate

In some embodiments, the substrates used in the present application aresilica-based substrates. In general, silica-based substrate surface ischemically modified in some way so as to attach covalently a chemicallyreactive group capable of reacting with the functionalized molecules,for example, hydrogel, polymer or a partially formed hydrogel (e.g. aprepolymer (PRP)). The surface-activating agent is typically anorganosilane compound. In one embodiment, the surface-activating agentis γ-methacryloxypropyltrimethoxysilane, known as “Bind Silane” or“Crosslink Silane” and commercially available from Pharmacia, althoughother silicon-based surface-activating agents are also known, such asmonoethoxydimethylsilylbutanal, 3-mercaptopropyl-trimethoxysilane and3-aminopropyltrimethoxysilane (all available from Aldrich). In this way,pendant functional groups such as amine groups, aldehydro groups orpolymerizable groups (e.g. olefins) may be attached to the silica.

The present application employs organosilane compounds comprisingcovalently attached cycloalkenes or heterocycloalkenes. In someembodiments, the cycloalkene is an optionally substituted norbornene. Insome embodiments, the silane moiety of the organosilane compounds hasthe following structure:

wherein R¹, R² and R³ are each independently selected from hydrogen,halogen, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted aryl, optionally substituted aryloxy, optionallysubstituted heteroaryl or optionally substituted heteroaryloxy. In somesuch embodiments, R¹, R² and R³ are independently optionally substitutedalkoxy. In some further embodiments, each of R¹, R² and R³ is methoxy.In one embodiment, the organosilane compound is[(5-bicyclo[2.2.1]hept-2-enyl)ethyl] trimethoxysilane.

Linker

In some embodiments described herein, the linker between the silane orsilane derivative and the cycloalkene or heterocycloalkene is selectedfrom an optionally substituted alkylene, an optionally substitutedheteroalkylene, an optionally substituted cycloalkylene, an optionallysubstituted heterocyclylene, an optionally substituted arylene, anoptionally substituted heteroarylene, an optionally substitutedpolyethylene glycol, a cleavable linker, or combination thereof.

In some embodiments, the linker described herein is an optionallysubstituted alkylene linker. In some embodiment, the linker is —(CH₂)n-,wherein n is selected from 1 to 20,000. In one embodiment, n is 2. Insome other embodiments, the linker described herein is an optionallysubstituted heteroalkylene linker. For example, the linker is —(CH₂)n-,wherein n is selected from 1 to 20,000, and one or more of the carbonatoms on the skeleton is replaced by one or more heteroatoms selectedfrom O, S, N or P.

In some embodiments, the linker described herein is a cleavable linker.In some embodiments, the linker is selected from acid labile linkers(including dialkoxybenzyl linkers, Sieber linkers, indole linkers,t-butyl Sieber linkers), electrophilically cleavable linkers,nucleophilically cleavable linkers, photocleavable linkers, cleavageunder reductive conditions, oxidative conditions, cleavage via use ofsafety-catch linkers, and cleavage by elimination mechanisms. In somesuch embodiments, L^(A) is selected from a disulfide linker (—S—S—),ester, nitrobenzene, imine, enzymatically or chemically cleavablepeptide and polynucleotide, such as DNA.

Cleavable linkers are known in the art, and conventional chemistry canbe applied to attach a linker to a nucleotide base and a label. Thelinker can be cleaved by any suitable method, including exposure toacids, bases, nucleophiles, electrophiles, radicals, metals, reducing oroxidizing agents, light, temperature, enzymes etc. The linker asdiscussed herein may also be cleaved with the same catalyst used tocleave the 3′-O-protecting group bond. Suitable linkers can be adaptedfrom standard chemical protecting groups, as disclosed in Greene & Wuts,Protective Groups in Organic Synthesis, John Wiley & Sons, or in Greg T.Hermanson's “Bioconjugate Techniques”, Academic Press. Further suitablecleavable linkers used in solid-phase synthesis are disclosed inGuillier et al. (Chem. Rev. 100:2092-2157, 2000).

The use of the term “cleavable linker” is not meant to imply that thewhole linker is required to be removed from, e.g., the reactiveheterocycle. As an example, the cleavage site can be located at aposition on the linker that ensures that part of the linker remainsattached to the heterocycle after cleavage.

A. Electrophilically Cleaved Linkers

Electrophilically cleaved linkers are typically cleaved by protons andinclude cleavages sensitive to acids. Suitable linkers include themodified benzylic systems such as trityl, p-alkoxybenzyl esters andp-alkoxybenzyl amides. Other suitable linkers includetert-butyloxycarbonyl (Boc) groups and the acetal system.

The use of thiophilic metals, such as nickel, silver or mercury, in thecleavage of thioacetal or other sulfur-containing protecting groups canalso be considered for the preparation of suitable linker molecules.

B. Nucleophilic Ally Cleaved Linkers

Nucleophilic cleavage is also a well-recognized method in thepreparation of linker molecules. Groups such as esters that are labilein water (i.e., can be cleaved simply at basic pH) and groups that arelabile to non-aqueous nucleophiles, can be used. Fluoride ions can beused to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethyl silane (TBDMS).

C. Photocleavable Linkers

Photocleavable linkers have been used widely in carbohydrate chemistry.It is preferable that the light required to activate cleavage does notaffect the other components of the modified nucleotides. For example, ifa fluorophore is used as the label, it is preferable if this absorbslight of a different wavelength to that required to cleave the linkermolecule. Suitable linkers include those based on O-nitrobenzylcompounds and nitroveratryl compounds. Linkers based on benzoinchemistry can also be used (Lee et al., J. Org. Chem. 64:3454-3460,1999).

D. Cleavage Under Reductive Conditions

There are many linkers known that are susceptible to reductive cleavage.Catalytic hydrogenation using palladium-based catalysts has been used tocleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction isalso known in the art.

E. Cleavage Under Oxidative Conditions

Oxidation-based approaches are well known in the art. These includeoxidation of p-alkoxybenzyl groups and the oxidation of sulfur andselenium linkers. The use of aqueous iodine to cleave disulfides andother sulfur or selenium-based linkers is also within the scope of thepresent application.

F. Safety-Catch Linkers

Safety-catch linkers are those that cleave in two steps. In a preferredsystem the first step is the generation of a reactive nucleophiliccenter followed by a second step involving an intra-molecularcyclization that results in cleavage. For example, levulinic esterlinkages can be treated with hydrazine or photochemistry to release anactive amine, which can then be cyclized to cleave an ester elsewhere inthe molecule (Burgess et al., J. Org. Chem. 62:5165-5168, 1997).

G. Cleavage by Elimination Mechanisms

Elimination reactions can also be used. For example, the base-catalyzedelimination of groups such as Fmoc and cyanoethyl, andpalladium-catalyzed reductive elimination of allylic systems, can beused.

In some embodiments, the linker can comprise a spacer unit. Otherexemplary suitable cleavable linkers are discussed in details in U.S.Publication No. 2006-0188901, which is hereby incorporated by referencein its entirety.

Sequencing Application

A method set forth herein can use any of a variety of amplificationtechniques. Exemplary techniques that can be used include, but are notlimited to, polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA), orrandom prime amplification (RPA). In particular embodiments, one or moreprimers used for amplification can be attached to a polymer coating. InPCR embodiments, one or both of the primers used for amplification canbe attached to a polymer coating. Formats that utilize two species ofattached primer are often referred to as bridge amplification becausedouble stranded amplicons form a bridge-like structure between the twoattached primers that flank the template sequence that has been copied.Exemplary reagents and conditions that can be used for bridgeamplification are described, for example, in U.S. Pat. No. 5,641,658;U.S. Patent Publ. No. 2002/0055100; U.S. Pat. No. 7,115,400; U.S. PatentPubl. No. 2004/0096853; U.S. Patent Publ. No. 2004/0002090; U.S. PatentPubl. No. 2007/0128624; and U.S. Patent Publ. No. 2008/0009420, each ofwhich is incorporated herein by reference. PCR amplification can also becarried out with one of the amplification primers attached to a polymercoating and the second primer in solution. An exemplary format that usesa combination of one attached primer and soluble primer is emulsion PCRas described, for example, in Dressman et al., Proc. Natl. Acad. Sci.USA 100:8817-8822 (2003), WO 05/010145, or U.S. Patent Publ. Nos.2005/0130173 or 2005/0064460, each of which is incorporated herein byreference. Emulsion PCR is illustrative of the format and it will beunderstood that for purposes of the methods set forth herein the use ofan emulsion is optional and indeed for several embodiments an emulsionis not used. Furthermore, primers need not be attached directly tosubstrate or solid supports as set forth in the ePCR references and caninstead be attached to a polymer coating as set forth herein.

RCA techniques can be modified for use in a method of the presentdisclosure. Exemplary components that can be used in an RCA reaction andprinciples by which RCA produces amplicons are described, for example,in Lizardi et al., Nat. Genet. 19:225-232 (1998) and US 2007/0099208 A1,each of which is incorporated herein by reference. Primers used for RCAcan be in solution or attached to a polymer coating.

MDA techniques can be modified for use in a method of the presentdisclosure. Some basic principles and useful conditions for MDA aredescribed, for example, in Dean et al., Proc Natl. Acad. Sci. USA99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003);Walker et al., Molecular Methods for Virus Detection, Academic Press,Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96 (1992); U.S. Pat.Nos. 5,455,166; 5,130,238; and 6,214,587, each of which is incorporatedherein by reference. Primers used for MDA can be in solution or attachedto a polymer coating.

In particular embodiments a combination of the above-exemplifiedamplification techniques can be used. For example, RCA and MDA can beused in a combination wherein RCA is used to generate a concatamericamplicon in solution (e.g. using solution-phase primers). The ampliconcan then be used as a template for MDA using primers that are attachedto a polymer coating. In this example, amplicons produced after thecombined RCA and MDA steps will be attached to the polymer coating.

In some embodiments, the functionalized hydrogel or polymer-coatedsubstrate described herein can be used for determining a nucleotidesequence of a polynucleotide. In such embodiments, the method cancomprise the steps of (a) contacting a polynucleotide polymerase withpolynucleotide clusters attached to a surface of a substrate via any oneof the polymer or hydrogel coatings described herein; (b) providingnucleotides to the polymer-coated surface of the substrate such that adetectable signal is generated when one or more nucleotides are utilizedby the polynucleotide polymerase; (c) detecting signals at one or morepolynucleotide clusters; and (d) repeating steps (b) and (c), therebydetermining a nucleotide sequence of a polynucleotide present at the oneor more polynucleotide clusters.

Nucleic acid sequencing can be used to determine a nucleotide sequenceof a polynucleotide by various processes known in the art. In apreferred method, sequencing-by-synthesis (SBS) is utilized to determinea nucleotide sequence of a polynucleotide attached to a surface of asubstrate via any one of the polymer coatings described herein. In suchprocess, one or more nucleotides are provided to a templatepolynucleotide that is associated with a polynucleotide polymerase. Thepolynucleotide polymerase incorporates the one or more nucleotides intoa newly synthesized nucleic acid strand that is complementary to thepolynucleotide template. The synthesis is initiated from anoligonucleotide primer that is complementary to a portion of thetemplate polynucleotide or to a portion of a universal or non-variablenucleic acid that is covalently bound at one end of the templatepolynucleotide. As nucleotides are incorporated against the templatepolynucleotide, a detectable signal is generated that allows for thedetermination of which nucleotide has been incorporated during each stepof the sequencing process. In this way, the sequence of a nucleic acidcomplementary to at least a portion of the template polynucleotide canbe generated, thereby permitting determination of the nucleotidesequence of at least a portion of the template polynucleotide. Flowcells provide a convenient format for housing an array that is producedby the methods of the present disclosure and that is subjected to asequencing-by-synthesis (SBS) or other detection technique that involvesrepeated delivery of reagents in cycles. For example, to initiate afirst SBS cycle, one or more labeled nucleotides, DNA polymerase, etc.,can be flowed into/through a flow cell that houses a nucleic acid arraymade by methods set forth herein. Those sites of an array where primerextension causes a labeled nucleotide to be incorporated can bedetected. Optionally, the nucleotides can further include a reversibletermination property that terminates further primer extension once anucleotide has been added to a primer. For example, a nucleotide analoghaving a reversible terminator moiety can be added to a primer such thatsubsequent extension cannot occur until a deblocking agent is deliveredto remove the moiety. Thus, for embodiments that use reversibletermination, a deblocking reagent can be delivered to the flow cell(before or after detection occurs). Washes can be carried out betweenthe various delivery steps. The cycle can then be repeated n times toextend the primer by n nucleotides, thereby detecting a sequence oflength n. Exemplary SBS procedures, fluidic systems and detectionplatforms that can be readily adapted for use with an array produced bythe methods of the present disclosure are described, for example, inBentley et al., Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No.7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492;7,211,414; 7,315,019; 7,405,281, and US 2008/0108082, each of which isincorporated herein by reference in its entirety.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568and 6,274,320, each of which is incorporated herein by reference in itsentirety). In pyrosequencing, released PPi can be detected by beingimmediately converted to adenosine triphosphate (ATP) by ATPsulfurylase, and the level of ATP generated can be detected vialuciferase-produced photons. Thus, the sequencing reaction can bemonitored via a luminescence detection system. Excitation radiationsources used for fluorescence based detection systems are not necessaryfor pyrosequencing procedures. Useful fluidic systems, detectors andprocedures that can be used for application of pyrosequencing to arraysof the present disclosure are described, for example, in WO 12/058096A1, US 2005/0191698 A1, U.S. Pat. Nos. 7,595,883, and 7,244,559, each ofwhich is incorporated herein by reference in its entirety.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference in its entirety. Some embodiments can includesequencing-by-hybridization procedures as described, for example, inBains et al., Journal of Theoretical Biology 135(3), 303-7 (1988);Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al.,Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which isincorporated herein by reference in its entirety. In bothsequencing-by-ligation and sequencing-by-hybridization procedures,nucleic acids that are present at sites of an array are subjected torepeated cycles of oligonucleotide delivery and detection. Fluidicsystems for SBS methods as set forth herein or in references citedherein can be readily adapted for delivery of reagents forsequencing-by-ligation or sequencing-by-hybridization procedures.Typically, the oligonucleotides are fluorescently labeled and can bedetected using fluorescence detectors similar to those described withregard to SBS procedures herein or in references cited herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andγ-phosphate-labeled nucleotides, or with zeromode waveguides (ZMWs).Techniques and reagents for FRET-based sequencing are described, forexample, in Levene et al. Science 299, 682-686 (2003); Lundquist et al.Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci.USA 105, 1176-1181 (2008), the disclosures of which are incorporatedherein by reference in its entirety.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in US 2009/0026082 A1; US2009/0127589 A1; US 2010/0137143 A1; or US 2010/0282617 A1, each ofwhich is incorporated herein by reference in its entirety.

Another useful application for an array of the present disclosure, forexample, having been produced by a method set forth herein, is geneexpression analysis. Gene expression can be detected or quantified usingRNA sequencing techniques, such as those, referred to as digital RNAsequencing. RNA sequencing techniques can be carried out usingsequencing methodologies known in the art such as those set forth above.Gene expression can also be detected or quantified using hybridizationtechniques carried out by direct hybridization to an array or using amultiplex assay, the products of which are detected on an array. Anarray of the present disclosure, for example, having been produced by amethod set forth herein, can also be used to determine genotypes for agenomic DNA sample from one or more individual. Exemplary methods forarray-based expression and genotyping analysis that can be carried outon an array of the present disclosure are described in U.S. Pat. No.7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Pat. Pub. Nos.2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which isincorporated herein by reference in its entirety.

In some embodiments of the above-described method which employ a flowcell, only a single type of nucleotide is present in the flow cellduring a single flow step. In such embodiments, the nucleotide can beselected from the group consisting of dATP, dCTP, dGTP, dTTP and analogsthereof. In other embodiments of the above-described method which employa flow cell, a plurality different types of nucleotides are present inthe flow cell during a single flow step. In such methods, thenucleotides can be selected from dATP, dCTP, dGTP, dTTP and analogsthereof.

Determination of the nucleotide or nucleotides incorporated during eachflow step for one or more of the polynucleotides attached to the polymercoating on the surface of the substrate present in the flow cell isachieved by detecting a signal produced at or near the polynucleotidetemplate. In some embodiments of the above-described methods, thedetectable signal comprises and optical signal. In other embodiments,the detectable signal comprises a non-optical signal. In suchembodiments, the non-optical signal comprises a change in pH at or nearone or more of the polynucleotide templates.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1 Immobilization of PAZAM on Norbornene 1a Silanized GlassSurface

Surface Silanization

Method 1 (silanization using glass vacuum desiccator): 200 μL-500 μL ofliquid norbornene silane were deposited inside a glass vial and placedinside a glass vacuum desiccator. Glass substrates were also placedinside the desiccator. The desiccator was then evacuated to a pressureof 15-30 mTorr, and placed inside an oven at a temperature between60-125° C. Silanization was let to proceed for 1 h, after which thedesiccator was removed from the oven, cooled and vented in air. Thesubstrates were utilized right after this step, or they were subjectedto an additional curing step (1 h at 100° C.) and/or a solvent washstep, such as an ethanol rinse.

Method 2 (silanization using YES CVD oven): First, substrates wereintroduced in the CVD oven chamber and the chamber was evacuated to apressure of 300 mTorr. The samples were initially treated with an oxygenplasma for 10 min. After plasma activation, the chamber was evacuated,and a 10 min rehydration cycle was executed by injecting 0.5 mL of waterat a base pressure of 500 mTorr. After an additional purging cycle, thesilanization program was executed. After a 15 mins delay time, thesilane valve was set to open for 0.15 sec, and to close for 20 sec.Silanization was performed at a base pressure of 500 mTorr and at achamber temperature of 125° C. for 60 min, and followed by 2 nitrogenpurge cycles, also at 125° C. The chamber was then vented over 3 min.The silanization cycle of the YES oven was highly automated andcontrolled. During the silanization step, the norbornene silane vesselwas kept at 120° C. and the silane vapor lines were kept at a constant125° C. The vacuum lines were kept at 145° C. After the cycle wascompleted, the substrates were removed, cooled outside the oven for abrief period and subsequently used without additional work-up. Thesesubstrates were viable for at least a month post-silanization.

PAZAM Deposition and Surface Crosslinking

500 μL of aqueous PAZAM (0.25%+5% ethanol) were deposited on top of anorbornene silanized glass substrate and spread across the surface. Athin film of PAZAM was obtained via spin coating with the followingprocedure: Step 1—600 rpm, 5 sec, acceleration 1500 rpm/sec; Step 2—1500rpm, 30 sec, acceleration 5000 rpm/sec; Step 3—4000 rpm, 5 sec,acceleration 5000 rpm/sec; Step 4—600 rpm, 5 sec, acceleration 5000rpm/sec. Other spin coat recipes can also be used. After spin-coating,the substrates were heated at 65-75° C. in oven or hot plate for 1 h.

Wash-Off: After the heating step, the substrates can be washed in waterto remove the unbound PAZAM by adding a sonication step (10 min) at 45°C., followed by extensive water rinse and drying with a nitrogen gun.

Primer grafting: the substrate prepared was used in the primer graftingstep by reacting alkyne oligonucleotides in KPi (10 mM) with PMDETA,copper sulfate and NaAsc (500 mg/mL aqueous solution) at 60° C. for 30minutes.

QC: After primer grafting step is completed, the grafted primers weresubjected to the TET quality control. TET is a dye labeledoligonucleotide having complimentary sequence to the P5/P7 primer. TETcan be hybridized to the P5/P7 primer on a surface; the excess TET canbe washed away, and the attached dye concentration can be measured byfluorescence detection using a scanning instrument such as a TyphoonScanner (General Electric). The intensity of the dye concentration wasmeasured as an indication of the percent surface remaining after thehydrogel immobilization. The PAZAM deposition and surface crosslinkingprocedures were also disclosed in U.S. application Ser. No. 13/784,368,which is incorporated by reference in its entirety.

FIG. 1A shows a D263 Schott glass substrate silanized with thenorbornene-silane derivative[(5-bicycle[2.2.1]hept-2-enyl)ethyl]trimethoxysilane (1a) andsubsequently coated and thermally cross-linked with PAZAM. The dark areais the actual fluorescence intensity observed upon grafting P5/P7primers and hybridizing with a TET-dye derivatized complementarystrands. FIG. 1B shows the related chart of median fluorescenceintensities for each lane of the same grafted norbornene (1a)silanized/PAZAM coated unpatterned surface hybridized with acomplimentary TET dye-containing oligonucleotide sequence.

Example 2 Preparation of Patterned Surface with PAZAM on NanowellSubstrate

Patterned, sequenceable clusters were created by integrating thenanowell substrates with the PAZAM polymer and chemical mechanicalpolishing (CMP). A nanowell substrate (400 nm diameter 750 nm pitch, 300nm depth well) was fabricated via a proprietary nanofabricationprocessed developed by Illumina and outsourced to Taiwan SemiconductorManufacturing Company Ltd (TSMC) using nanoimprint lithography.Norbornene silane of Example 1 was deposited by CVD on the entiresurface of the substrate and PAZAM was spin coated and heated at 60-70°C., creating a covalent linkage of the polymer to the substrate surface.The interstitial covalently linked polymer was removed by polishing thesurface with 10 wt % 3 μm SiO₂ micro particle slurry in water, throughthe CMP process. The patterned polymer substrate was then grafted withprimers following standard Illumina protocol. The patterned primers onthe substrate were imaged with a Typhoon imager. The substrate was thenseeded with phiX DNA, clustered with Illumina's proprietaryamplification protocol, derived from the Twist DX kit (isothermalamplification) and sequenced. Sequencing was conducted on an IlluminaHiSeq 2000, using the standard SBS sequencing reagent kit and themetrics were extracted using Illumina's sequencing analysis viewer. Thesequencing analysis data showed that the sequencing metrics of thenorbornene silanized substrate run are equivalent to those of substratesfunctionalized with acrylamides.

FIG. 2A shows a D263 Schott glass substrate patterned with nanowellssilanized with the norbornene-silane derivative (1a) and subsequentlycoated and thermally cross-linked with PAZAM. The dark area is theactual fluorescence intensity observed after chemical mechanicalpolishing of the excess PAZAM and upon grafting P5/P7 primers andhybridizing with a TET-dye derivatized complementary strands. FIG. 2Bshows the related chart of median fluorescence intensities for each laneof the same grafted norbornene (1a) silanized/PAZAM coated unpatternedsurface hybridized with a complimentary TET dye-containingoligonucleotide sequence.

Not only did norbornene silanized substrate eliminate the need ofadditional cross-linking agent, norbornene silanized substratesdisplayed a preferential affinity for PAZAM and facilitated thespreading of aqueous PAZAM solution. As a result, the PAZAM coatingswere more homogeneous and less sensitive to variations in substratequality. FIG. 3A shows the typhoon image of a flow cell as a result oftypical failure of spin coating using standard acrylamidesfunctionalized surface. FIG. 3B shows the typhoon image of a norbornenesilane derivative (1a) silanized flow cell, which results in morehomogeneous coating. FIG. 3C shows that a 0.25% aqueous PAZAM solutionwets a norbornene silane derivative (1a) silanized surface in comparisonwith water smears, even in the presence of a large energy mismatchbetween surface energies of the solid-liquid interface.

Example 3 Surface Stability Testing

Norbornene silanized/PAZAM coated substrates also demonstrated goodshelf life. Several patterned substrates and un-patterned substrateswere silanized with norbornene silane of Example 1 using YES method,then subject to PAZAM coating and crosslinking using the methoddescribed in Exampled 1 and 2. The patterned substrates were stored in aslide carrier in the dark at room temperature, inside a desiccator.After 30 days, these substrates were sequenced and provided acceptableTET QC results and sequencing metrics (FIG. 4).

Example 4 Primer Grafting with BCN Modified-Oligos

One embodiment of a method for grafting BCN-modified oligos to aPAZAM-coated surface is as follows: 5′-modified BCN P5 and P7 oligoscontaining the same sequences as the standard P5 and P7 (thiophosphateand alkyne) oligos were used to react with a flow cell surface coatedwith PAZAM (0.25% w/v) without any catalysts (Scheme 2). The PAZAMcoated surface was prepared by first treating the surface with3-aminopropyltrimethoxysilane (APTMS), followed by treatment withacryloyl chloride (80 μL of neat acryloyl chloride and 40 μL of DIPEA in1880 μL anhydrous MeCN) or activated acryloyl NHS ester (20 mg/mL in KPipH=8.0, 200 mM) to form the unsaturated acrylamide groups. Then, PAZAMwas introduced to the unsaturated surface and the substrate wasincubated at 60° C. for 50-75 min (static). The general method isdescribed in U.S. application Ser. No. 13/784,368, which is herebyincorporated by reference in its entirety. The detailed experimentalconditions for each lane of the flow cell are illustrated in Table 1below.

TABLE 1 Total Details [alkyne]/uM 1% PAZAM, standard Blackpool grafting2 1% PAZAM, standard Blackpool grafting 2 1% PAZAM, Copper-freegrafting, P5 and P7 BCN primers 2 1% PAZAM, Copper-free grafting, P5 andP7 BCN primers 2 1% PAZAM, Copper-free grafting, P5 and P7 BCN primers 21% PAZAM, Copper-free grafting, P5 and P7 BCN primers 2 1% PAZAM,Copper-free grafting, P5 BCN primer only 2 1% PAZAM, Copper-freegrafting, P7 BCN primer only 2

An initial Typhoon image of the grafted surface of the flow cell usingthe catalyst-free grafting of BCN modified oligos described above isdepicted in FIG. 5A. The flow cell surface was subjected to a thermalStress test to determine the robustness of the grafted coating and theresult shown in FIG. 5B demonstrated that the signal reduction(approximately correlated with surface loss) is minimal and consistentwith the standard lanes.

To explore the experimental condition for obtaining optimal primerdensities, different concentrations of BCN modified oligos were alsotested (Table 2). The PAZAM coated flow cell surface was prepared from anorbornene modified silane using the similar procedure described inExample 1. Then, different concentrations of BCN modified primers weregrafted to the polymer layer. The initial Typhoon image of the graftedsurface of a flow cell using copper-free grafting method and differentconcentrations of BCN primers is demonstrated in FIG. 6A. The flow cellsurface was also subjected to a thermal Stress test and FIG. 6Bindicates the results from TET QC analyses performed before and aftersurface thermal stressing.

TABLE 2 Channel Details Total [alkyne]/uM 1 0.25% PAZAM, Copper-freegrafting 2 2 using P5 and P7 BCN primers 2 3 4 4 4 5 10 6 10 7 20 8 20

Bridge amplification performed on both grafted flow cell surfacesproceeded smoothly. The clusters were viewed using a fluorescencemicroscope, and they appeared comparable with those grown in the controllanes. FIG. 7A shows a fluorescence image of clusters grown from a BCNprimer grafted surface. The template seeding concentration was 0.5 pM.Similarly, FIG. 7B shows fluorescence images of clusters grown from aBCN primer grafted surface where the template concentration was 3 pM.

The BCN modified oligos-grafted flow cell surfaces were then takenthrough several sequencing runs using a HiSeq instrument. The high levelmetrics were comparable with the control lanes. Comparison of the SBSdata shows that the high-level sequencing metrics are very comparablewith results currently obtained from PAZAM surfaces grafted usingstandard alkyne oligos.

Example 5 Primer Grafting with BCN-Oligos

In some embodiments of the substrate surface preparation processesdescribed herein, the raw substrates are first coated with a silanederivative, e.g., norbornene derivatized silane, then PAZAM is spincoated on to the surface. The substrate is then polished and assembled,subsequently subjected to primer grafting and QC. The alternativeapproach of preparing substrate using pre-grafted PAZAM byfunctionalizing PAZAM with the standard P5 and P7 oligos in solution wasalso explored. This process offers several important advantages. Movingthe primer grafting step upstream would allow for more effective polymerpurification and greater control of the amount of oligos used to achievea target surface primer density. In addition, the workflow to a finishedsubstrate can be shortened as the coated substrate surface would alreadycontain the primers required for template hybridization and thusremoving the need to use specialized fluidic instrumentation to achieveadequately grafted surfaces.

A solution of the mixed P5/P7 BCN modified primers (totalconcentration=15 μM) was added to an aqueous solution of PAZAM (0.5 w/v%) and the resulting mixture was heated for 2 hours at 70° C. Aftercooling to room temperature, the mixture was used to coat a standardHiSeq flowcell, pre-treated with a norbornene-derivatized silane layer(FIG. 11).

The pre-grafted surface was subjected to the standard thermal stresstest and the signal losses (corresponding to surface losses) werecomparable to the control. A 2×26 HiSeq SBS sequencing experiment wasperformed using the standard reagents as described in Illumina standardprotocol. FIG. 8A shows the type of clusters obtained from a pre-graftedPAZAM surface. The seeding template concentration was reduced (0.5 pM).In FIG. 8B, the bottom surface from a channel in the same flowcell isshown with the seeding concentration increased to 3 pM. The change incluster density is within expected limits and therefore shows that thisnew surface behaves very similarly to control surfaces. Inspection ofthe images (FIGS. 8A and 8B) indicated that the clusters were typicalwith the only variation being the apparent cluster “size” as estimatedby consideration of the Full Width Half Max (FWHM), which is usedinternally as a proxy for the apparent cluster size. This is consistentwith the reduced intensity of these clusters. A comparison between theSBS metrics for the pre-grafted, control and BCN modified primer-graftedchannels confirms that SBS cycles were proceeding from the clustersgenerated on the surface.

A similar, longer SBS run was performed using a MiSeq instrument. Inthis case, a standard PAZAM solution was incubated with the BCN-modifiedprimers for 3 hours at 70° C. The resulting grafted polymer mixture wasthen applied to a norbornene functionalized MiSeq flowcells followingthe standard protocols. On-board cluster generation was followed by a2×151 cycle SBS run performed using a standard four-channel system. FIG.9 shows a thumbnail image of clusters grown from a channel surfacecoated with pre-conjugated PAZAM mixture obtained from a standard MiSeqsystem (2×151 SBS). The right portion of the image is a magnification ofthe left portion of the image.

The SBS images for all cycles from this run are comparable with thosefrom standard (SFA and PAZAM) SBS experiments. The signal-to-noisemeasurements are also very similar compared against standard surfaces,showing that the no obvious entrapment of dye molecules takes placeduring sequencing (i.e. the polymer coating does not appear to undergoadditional changes).

What is claimed is:
 1. A method of grafting primers on a first surfaceof a substrate, the method comprising: providing pre-conjugated primerscomprising oligonucleotides covalently bonded to a functionalizedpolymer or hydrogel, wherein the functionalized polymer or hydrogelcomprises functional groups; and contacting the pre-conjugated primerswith a substrate having a first surface comprising a silane or a silanederivative, wherein the silane or silane derivative comprises a firstplurality of unsaturated moieties selected from the group consisting ofcycloalkenes, cycloalkynes, heterocycloalkenes, heterocycloalkynes, andoptionally substituted variants and combination thereof covalentlyattached thereto, and wherein the pre-conjugated primers are covalentlybound to the first surface of the substrate through the reaction of thefunctional groups of the functionalized polymer or hydrogel with thefirst plurality of unsaturated moieties of the silane or silanederivative; wherein the functionalized polymer or hydrogel comprises arecurring unit of Formula (I) and a recurring unit of Formula (II):

wherein R¹ is H or alkyl; R^(A) is selected from the group consisting ofazido, optionally substituted amino, optionally substituted alkenyl,optionally substituted hydrazone, optionally substituted hydrazine,carboxyl, hydroxy, optionally substituted tetrazole, optionallysubstituted tetrazine, nitrile oxide, nitrone, and thiol; X is anoptionally substituted alkylene or an optionally substitutedheteroalkylene; R⁴, R^(4′), R⁵ and R^(5′) are each independentlyselected from the group consisting of H, R⁶, OR⁶, —C(O)OR⁶, —C(O)R⁶,—OC(O)R⁶, —C(O)NR⁷R⁸, and —NR⁷R⁸; R⁶ is independently selected from thegroup consisting of H, OH, alkyl, cycloalkyl, hydroxyalkyl, aryl,heteroaryl, and heterocyclyl, and optionally substituted variantsthereof; and R⁷ and R⁸ are each independently H or alkyl, or R⁷ and R⁸are joined together with the atom or atoms to which they are attached toform a heterocycle.
 2. The method of claim 1, wherein the pre-conjugatedprimers are prepared by reacting the functional groups of thefunctionalized polymer or hydrogel with a second plurality ofunsaturated moieties of the oligonucleotides to form covalent bonding,and wherein the second plurality of unsaturated moieties of theoligonucleotide are selected from the group consisting of alkynes,cycloalkenes, cycloalkynes, heterocycloalkenes, and heterocycloalkynes,and optionally substituted variants and combinations thereof.
 3. Themethod of claim 1, wherein the first plurality of unsaturated moietiesare selected from the group consisting of norbornene, heteronorbornenes,norbornene derivatives, trans-cyclooctene, trans-cyclooctenederivatives, cyclooctyne, and bicycloalkynes, and optionally substitutedvariants and combinations thereof.
 4. The method of claim 1, wherein thefirst plurality of unsaturated moieties are optionally substitutednorbornene.
 5. The method of claim 2, wherein the second plurality ofunsaturated moieties are selected from the group consisting ofoptionally substituted cyclooctyne, optionally substitutedbicyclononynes, and optionally substituted bicyclo[6.1.0]non-4-yne. 6.The method of claim 1, wherein the silane or silane derivative areapplied onto the first surface by chemical vapor deposition or YieldEngineering Systems (YES) method.
 7. The method of claim 1, wherein thefunctional groups of the functionalized polymer or hydrogel compriseazido, optionally substituted amino, optionally substituted alkenyl,optionally substituted hydrazone, optionally substituted hydrazine,carboxyl, hydroxy, optionally substituted tetrazole, optionallysubstituted tetrazine, nitrile oxide, nitrone, or thiol, or combinationsthereof.
 8. The method of claim 7, wherein the functional groups of thefunctionalized polymer or hydrogel comprise azido.
 9. The method ofclaim 1, wherein R⁴ is —C(O)NH₂; R⁵ is H or alkyl; each R^(4′) andR^(5′) is H; X is optionally substituted —(CH₂)_(p)—; and p is aninteger in the range of 1 to
 50. 10. The method of claim 9, wherein p is5 and R^(A) is azido.
 11. The method of claim 9, wherein both R¹ and R⁵are H.
 12. The method of claim 1, wherein the first plurality ofunsaturated moieties is covalently attached to the silicon atoms of thesilane or silane derivative through a linker.
 13. The method of claim12, wherein the linker is selected from the group consisting ofoptionally substituted alkylenes, optionally substitutedheteroalkylenes, optionally substituted cycloalkylenes, optionallysubstituted heterocyclylenes, optionally substituted arylenes,optionally substituted heteroarylenes, optionally substitutedpolyethylene glycols, cleavable linkers, and combinations thereof. 14.The method of claim 12, wherein the linker is selected from the groupconsisting of optionally substituted alkylenes and optionallysubstituted heteroalkylenes.
 15. The method of claim 14, wherein thelinker is an optionally substituted alkylene.
 16. The method of claim 1,further comprising a washing step to remove excess unboundedpre-conjugated primers.
 17. The method of claim 1, further comprising awashing step to remove excess unbounded oligonucleotides.
 18. The methodof claim 1, further comprising a drying step.